Polyhydroxyalkanoate levels as an indicator of bioreactor health

ABSTRACT

A method has been developed to monitor the health of an activated sludge environment in a wastewater process comprising monitoring the levels of polyhydroxyalkanoates (PHA) produced and correlating those levels with various selected sample parameters. In general, levels of PHA in excess of about 15% to about 20% dry weight of the biomass is an indication that the biocatalytic efficiency of the wastewater treatment process is impaired.

This application claims the benefit of U.S. Provisional Application No.60/231,025 filed Sep. 8, 2000.

FIELD OF THE INVENTION

The present invention relates to methods for monitoring and controllingbiological activity in wastewater and controlling the treatment thereof.Specifically a method has been developed that correlates the productionof polyhydroxyalkanoates (PHA) with bioreactor health and biocatalyticefficiency.

BACKGROUND OF THE INVENTION

A number of devices and systems to process and purify water fromindustrial operations and municipal sources prior to discharging thewater are known. Activated-sludge wastewater treatment plants, which arewell known in the art, have been most often utilized to address thisproblem. Additionally, many industrial and municipal water treatmentplants utilize biological systems to pre-treat their wastes prior todischarging into the usual municipal treatment plant. In theseprocesses, the microorganisms used in the activated sludge break down ordegrade contaminants for the desired water treatment. Efficient processperformance and control requires quick and accurate assessment ofinformation on the activity of microorganisms. This has proven to be adifficult task in view of the wide variety of materials and contaminantsthat typically enter into treatment systems. Variations in the quantityof wastewater being treated, such as daily, weekly or seasonal changes,can dramatically change numerous important factors in the treatmentprocess, such as pH, temperature, nutrients and the like, the alterationof which can be highly detrimental to proper wastewater treatment.Improperly treated wastewater poses serious human health dangers. It isimperative therefore to maintain the health and biocatalytic efficiencyof these activated sludge systems.

Various biological processes are currently used in wastewater treatmentplants to assist in contamination degradation. In a typical process,contaminants in the wastewater, such as carbon sources (measured asbiological oxygen demand or BOD), ammonia, nitrates, phosphates and thelike are digested by the activated sludge in anaerobic, anoxic andaerobic stages, also known in the art. In the anaerobic stage, thewastewater, with or without passing through a preliminary settlementprocess, is mixed with return activated sludge.

The goal of wastewater bioreactors is to mineralize inlet organic andinorganic compounds (nitrogen oxides e.g., nitrate, nitrite, andammonia) to carbon dioxide and nitrogen gas resulting in a cleaneffluent stream. The efficiency of industrial wastewater treatmentsystems is especially important since loss of performance/capacity totreat process wastewater translates to lower manufacturing up time.Presently, there are no rapid methods to assess the biocatalyticcapacity of wastewater reactors.

Currently, crude macroscopic parameters are used to gauge theperformance of wastewater bioreactors. These crude macroscopicparameters include: exit carbon as measured by COD (chemical oxygendemand) or TOC (Total Organic Carbon); exit nitrogen by Total KjeldahlNitrogen (TKN) and exit phosphate by Ion Chromotography. Whilemeasurements of effluent leakage of exogenously supplied carbon, such asmethanol and organic acid, could be used to identify an impairedbioreactor, these compounds would not be reliable indicators ofbioreactor health because many other processes can impact the amount ofcarbon removed. Furthermore, assessment of performance using thesemetrics results in a responsive operating strategy, i.e., changes toreactor loads that are made only after deviation from the desiredperformance is observed. Finally, the catalytic activity of the biomasspresent in the bioreactors is time consuming to measure. For example,the denitrification rate is determined by removing biomass from thereactor and performing a batch rate study; taking about 1-2 days toperform, and therefore cannot be used to gauge the currentdenitrification capacity of the reactor. These batch studies are usefulin establishing long term performance characteristics. To date, therehave been no reports describing the relationship between cell physiologyand catalytic capacity wastewater bioreactors.

The above methods are useful for monitoring the health of activatedsludge systems however they contain several drawbacks including theinability to accurately predict a reduction or loss of denitrificationactivity in the system before nitrate or one of the denitrificationintermediates is present in the bioreactor. This results in nitrateleakage or incomplete denitrification that is highly detrimental andundesirable to such systems. An improved method of tracking biocatalyticefficiency is needed, particularly with respect to denitrificationpotential.

The problem to be solved, therefore is to provide a facile, highlyresponsive method of monitoring activated sludge environments to rapidlypredict loss of denitrification activity and other indicators ofbiocatalytic efficiency such as the concentrations of nitrate, ammonia,sulfate, phosphate and carbon dioxide in the system.

SUMMARY OF THE INVENTION

The present invention provides reliable methods to monitor bioreactorhealth and to maintain viable cultures within the bioreactor.Specifically, Applicants have solved the above-stated problem by makingthe correlation between the production of an internal storage moleculeand denitrification rate as a control strategy. This internal storagemolecule may be a class of storage molecules, collectively termedpolyhydroxyalkanoates (PHA), or glycogen or the like. Preferably, thisinternal storage molecule is polyhydroxyalkanoates (PHA). The PHA levelin the bacteria of the activated sludge can be easily measured andcontrolled by the level of nutrients (ammonia, phosphate, sulfate) inthe bioreactor.

Specifically, the present invention provides a method for monitoring andcontrolling the biocatalytic efficiency of a wastewater treatmentprocess comprising: a) providing an activated sludge environmentcomprising:

(i) a carbon influx;

(ii) cultures of autotrophic, heterotrophic and facultativemicroorganisms;

(iii) feed nutrients; and

(iv) an end electron acceptor; b) sampling wastewater from anaerobic,anoxic and/or aerobic stages of the treatment process; c) measuring theconcentration of polyhydroxyalkanoates present in the sample todetermine the status of selected sample characteristics; and d)adjusting the feed nutrients in the activated sludge environmentdepending on the status of the selected sample as measured in c),whereby the biocatalytic efficiency of a wastewater treatment process iscontrolled.

An indication that the biocatalytic efficiency of the wastewatertreatment process is impaired is seen when the polyhydroxyalkanoatesconcentration is greater than about 15 to about 20 dry weight percent ofthe biomass. Preferably, the feed nutrients are adjusted accordingly inthe activated sludge environment when the PHA concentration is fromabout 10 to about 20 dry weight percent of the biomass. More preferably,the feed nutrients are adjusted accordingly in the activated sludgeenvironment when the PHA concentration is from about 10 to about 15 dryweight percent of the biomass.

Sample characteristics are selected from the group consisting ofefficiency of denitrification, nitrate concentration, ammoniaconcentration, sulfate concentration, phosphate concentration and carbondioxide concentration. Similarly, feed nutrients are selected from thegroup consisting of nitrate, ammonia, sulfate, sulfide, urea andphosphate. In addition, an end electron acceptor is selected from thegroup consisting of oxygen, nitrate, nitrite, nitrous oxide, ferricoxide, and sulfate.

The invention additionally provides a method of maintaining viablecultures in an activated sludge environment in the absence of carboninflux comprising: a) providing an activated sludge environmentcomprising:

(i) a carbon influx;

(ii) cultures of autotrophic, heterotrophic and facultativemicroorganisms;

(iii) feed nutrients; and

(iv) an end electron acceptor; b) removing the feed nutrients from theactivated sludge environment while continuously monitoring theconcentration of polyhydroxyalkanoates present in the activated sludgeenvironment; c) removing the carbon influx from the activated sludgeenvironment when the concentration of polyhydroxyalkanoates is greaterthan about 15 to about 20 dry weight percent of the biomass; and d)adding a minimal concentration of nitrate to the activated sludgeenvironment of step c); whereby the cultures of autotrophic,heterotrophic and facultative microorganisms are maintained in a viablestate in the absence of a carbon influx.

In a preferred embodiment, the carbon influx is removed from theactivated sludge environment when the concentration of PHA is about 20dry weight percent of biomass or greater.

In another preferred embodiment, the minimal concentration of nitrateadded to the activated sludge environment of step c) is based on the CODcontent of the PHA in the biomass. Specifically, the COD provided by thePHA is equal to the fraction of PHA times the total dry biomass timesfraction of carbon in PHA times the conversion factor for TOC to COD andis represented by Equation 1.

PHA COD(mg/L)=(0.2 mg PHA/mg MLSS)×[MLSS(mg/L)]×(0.556 mg C/mgPHA)  EQUATION 1:

The amount of nitrate that can be added to the activated sludgeenvironment is equal to the COD provided by the PHA multiplied by theamount of nitrate-N reduced/COD)×(4.5 mg nitrate/mg nitrate-N).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Macroscopic Reactor Performance without Ammonia. Nitrateload in mg/d (closed circle), Nitrate exiting reactor in mg/L (squares).Lines are drawn to indicate data trends. Arrow 1: Switch from methanolrich feed to organic acids rich feed. Arrow 2: First reactor shut down.Arrow 3: Second reactor shut down.

FIG. 2 shows the Reactor Performance over Time in days without Ammonia.2A: NUR vs. time with organic acid rich feed composition as carbonsource. (Arrow 1: Switch from methanol rich feed to organic acids richfeed. Arrow 2: First reactor shut down. Arrow 3: Second reactor shutdown.) 2B: NUR vs. time with methanol as carbon source. 2C: No carboncontrol. 2D: PHA concentration in percent biomass dry weight. NUR=mgnitrate-N/mg MLVSS-min.

FIG. 3 shows the correlation between Nitrate Uptake Rate (NUR) and PHALevel. NUR with organic acid rich feed composition as carbon source(closed circles). NUR with methanol as carbon source (X). The hatchedline represents a linear regression of the organic acid rich feedcomposition data.

FIG. 4 shows the Macroscopic Reactor Performance with Ammonia Addition.Nitrate load in mg/d (closed circle), Nitrate exiting reactor in mg/L(open square). Lines are drawn to illustrate data trends.

FIG. 5 shows the Reactor Performance over Time with Ammonia Addition.5A: NUR vs. time with organic acid rich feed composition as carbonsource. 5B: NUR vs. time with methanol as carbon source. 5C: No carboncontrol. 5D: PHA concentration in percent biomass dry weight. NUR=mgnitrate-N/mg MLVSS-min.

FIG. 6 shows the internal PHA as a carbon source. Closed circlesrepresent PHA weight fraction of the cell mass. Closed squares representthe nitrate concentration in the reactor. Lines are drawn to indicatethe trends.

FIG. 7 shows the PHA accumulation during ammonia limitation. Squaresrepresent the nitrate load to the reactor, circles represent theresidual nitrate in the reactor and diamonds represent the weightpercent of PHA in the biomass.

The invention can be more fully understood from the following detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is useful for monitoring and controllingbioreactor health and bioreactor catalytic efficiency in an activatedsludge environment by correlating the level of denitrification in thesludge with the production of an internal storage molecule. Thisinternal storage molecule may be polyhydroxyalkanoates (PHA), glycogen,or the like. Preferably, this internal storage molecule ispolyhydroxyalkanoates (PHA).

The method of the invention comprises providing an activated sludgeenvironment that comprises a number of elements including a carboninflux, a variety of autotrophic, heterotrophic and facultativemicroorganisms, feed nutrients and at least one type of end electronacceptor to support respiration by the heterotrophic bacteria.Wastewater from the activated sludge system is then sampled from any ofthe anaerobic, anoxic, and/or aerobic stages of the bioreactor and theconcentration of the internal storage molecule(s) is measured in thesample's bacteria according to methods well known in the art. Apreferred method of measuring PHA levels is the method described by Riisand Mai, 1988 (Journal of Chromatography, 445:285-289) as outlined infrain the General Methods section of the Examples. Glycogen levels can bedetermined according to the methods described within Gerhardt et al.(Manual of Methods for General Bacteriology, American Society forMicrobiology, Washington, D.C., 1981). Levels of PHA in excess of15%-20% dry weight percent of the biomass indicate that the biocatalyticefficiency of the wastewater treatment process is impaired, and thatdenitrification potential in particular may be compromised. Feednutrients may be adjusted accordingly to reverse the imbalance.Preferably, the feed nutrients are adjusted accordingly in the activatedsludge environment when the PHA concentration is from about 10 to about20 dry weight percent of the biomass. More preferably, the feednutrients are adjusted accordingly in the activated sludge environmentwhen the PHA concentration is from about 10 to about 15 dry weightpercent of the biomass.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

The term “biocatalytic efficiency” will mean the specific removal rateof the target compound e.g., grams of organic removed per grams ofbiomass per time. Biocatalytic efficiency is generally consideredimpaired when the levels of PHA within the biomass are in excess ofabout 15 to about 20 dry weight percent of the biomass.

The term “denitrification” relates to a biological anaerobic processthat occurs under reduced conditions in which nitrate acts as theelectron acceptor to convert nitrate or nitrite to gaseous products.Many bacteria are able to perform this denitrification process to firstreduce nitrate to nitrite, and then nitrite to nitric oxide (NO),nitrous oxide (N₂O), and finally nitrogen gas (N₂). Within a wastewateractivated sludge environment, denitrification or nitrate reduction is akey process that is required to effectively treat wastewater prior toeffluent release.

The term “denitrification efficiency” will mean the extent of reductionof nitrate or nitrite to the desired end product dinitrogen (N₂).

The term “denitrification potential” will mean the rate of reduction ofnitrate to dinitrogen by a unit of biomass.

The term “wastewater treatment process” will mean a biological reactorsystem comprising an activated sludge environment that has the abilityto degrade various organic compounds and other persistent pollutants. Asused herein the term “activated sludge environment” will mean themixture of wastewater, bacteria, and nutrients needed to maintain thehealth of the bacteria and effect the biocatalytic degradation oforganic molecules.

The term “carbon influx” will mean the composition of organic materialentering into a wastewater treatment process. The carbon influx willtypically contain a variety of aromatic and straight chain organicmolecules from industrial processing streams. With regard to thecomposition of organic material entering into a wastewater treatmentprocess as carbon influx, this influx may comprise organic compoundsselected from the group consisting of amines, alcohols, organic acids,carbohydrates, proteins, and amino acids.

The term “selected sample characteristics” refers to the characteristicsof a wastewater sample that is tested for PHA concentrations. Selectedsample characteristics typically define the health of a wastewatersystem and will relate to concentrations of various nutrients, includingbut not limited to nitrate, ammonia, sulfate, sulfide, urea, phosphateand carbon dioxide.

The term “feed nutrients” means various nutrients necessary to maintainthe health and biocatalytic efficiency of the activated sludgeenvironment. The components that comprise a wastewater system generallyinclude an organic source (e.g., carbohydrates, organic acids andalcohols, amino acids, peptides, and/or aromatics), an end electronacceptor (such as oxygen, nitrate, nitrite, nitrous oxide, sulfate,and/or ferric iron), and “feed nutrients” (sulfate, sulfide, phosphate,and a nitrogen source, such as nitrate, ammonia, and/or urea). Theorganic source supplies energy and carbon molecules for anabolism. Theend electron acceptor functions as the molecule that is reduced duringenergy generation. Feed nutrients are needed as building blocks formacromolecule synthesis of cellular constituents such as amino acids,deoxyribonucleotides, ribonucleotides, and phospholipids. Preferably,more than one nutrient is present in the feed nutrients. In a specificembodiment of the invention, the feed nutrients comprise free ammonia, asulfate, and potassium phosphate or ammonium phosphate as nutrients.

The term “end electron acceptor” will mean a molecule that is reduced asa result of microbial respiration. Examples include, but are not limitedto, oxygen, nitrate, nitrite, nitrous oxide, ferric oxide, and sulfate.

The term “nitrate-N” will mean the amount of nitrogen present per moleof nitrate. The conversion factor is 4.5 g nitrate/g nitrogen.Therefore, 20 mg/L nitrate-N is equal to 90 mg/L nitrate.

The term “mixed liquor suspended solids” or “MLSS” refers to the dryweight of the biomass in the system and is measured by drying tocompletion a known volume from the bioreactor at 105° C.

The term “mixed liquor volatile suspended solids” or “MLVSS” refers tothe weight of volatile suspended solids in the system and is determinedby heating the dried sample at 550° C. for 30 min, followed by coolingto room temperature in a dessicator, and measuring the weight. “MLVSS”is calculated by subtracting the residual weight of the sample followingthe 550° C. heating step from “MLSS” weight.

The terms “activated sludge bacteria”, “activated sludge microorganisms”or “sludge” will refer to autotrophic, heterotrophic and facultativemicroorganisms that are typically found in wastewater systems andpossess the enzymatic machinery to degrade compounds found in carboninflux.

Activated Sludge Environment

The present invention provides an activated sludge environmentcomprising a number of components including a carbon source, a varietyof autotrophic, heterotrophic, and/or facultative bacteria, and feednutrients required for the growth and biocatalytic health of thebiomass. Preferably, all three types (autotrophic, heterotrophic andfacultative) of bacteria are present.

The term “autotrophic bacterium” or “autotrophic bacteria” will mean anorganism or organisms capable of growing on inorganic nutrients andusing carbon dioxide as its sole carbon source.

The term “heterotrophic bacterium” or “heterotrophic bacteria” will meanan organism or organisms that requires one or more organic nutrients,including an organic carbon source for growth, e.g., the requirement ofglucose by Escherichia coli for growth.

The term “facultative bacterium” or “facultative bacteria” will mean anorganism or organisms that can switch easily from one growth physiologyto another. For example, bacteria that can switch to nitrate as anelectron acceptor in the absence of oxygen.

The types of bacteria that may be present in the activated sludgeenvironment include, but are not limited to, Proteobacteria, aphysiologically diverse group of microorganisms that represents fivesubdivisons (α, β, γ, ε, δ), (Madigan et al. Biology of Microorganisms,8^(th) ed. Prentice Hall Upper Saddle River, N.J. 1997). Specificexamples of bacterial genera that are expected to work or be involved inthe activated sludge environment include, but are not limited to,Paracoccus, Rhodococcus, Pseudomonas, Alcaligenes, Acinetobacter,Sphingamonas, Azoarcus, and Burkholderia.

Internal Storage Molecules

The production of an internal storage molecule is used within themethods of the present invention to monitor and control bioreactorhealth, to predict and assess bioreactor catalytic efficiency, and tomaintain an activated sludge environment. This internal storage moleculemay be a class of molecules, collectively termed polyhydroxyalkanoates(PHA), or glycogen, or the like. Both PHA and glycogen have beenextensively studied as energy-storage compounds (see Dawes and Senior,1973. Adv. Microb. Physiol. 10:135-266) but their regulation is notclearly understood. In a preferred embodiment of the present invention,this internal storage molecule is polyhydroxyalkanoates (PHA).

Polyhydroxyalkanoates

The terms “polyhydroxyalkanoates” or “PHA” are used herein as genericterms for a class of molecules that are primarily linear, head-to-tailpolyesters composed of 3-hydroxy fatty acid monomers that accumulate inmicrobes as carbon and energy storage molecules (see Madison andHuisman, 1999. Microbiol. Mol. Biol. Rev., 63:21-53; and Byrom, 1994.In: Mobley, ed. Plastics from Microbes, Hanser Publishers, New York, pp.5-33 for reviews). These polymers are generally synthesized in a broadrange of bacteria when the cells have adequate carbon supplies but arelimited for another nutrient, such as nitrogen, phosphate, or oxygen.

The terms “polyhydroxyalkanoates” or “PHA” refer to a class of compoundsof the general formula:

where R═CH₃ or CH₃(CH₂)_(m), where m=1 to 10, and n=4,000 to 20,000(Mobley, D, editor. Plastics From Microbes. Hanser Publishers, New York,1994).

The most commonly occurring PHA molecules are polyhydroxybutyrate (PHB)and polyhydroxyvalerate/polyhydroxybutyrate co-polymer that results froma condensation of two molecules of acetyl CoA to form 3-hydroxybutyrylCoA or acetyl CoA with propionyl CoA to form 3-hydroxyvaleryl CoA. Theseactivated molecules are then incorporated into the polymer.

In addition to the 3-hydroxyalkonoates described above, some bacteriahave the ability to incorporate longer chained 3-hydroxy acids (carbonlength up to C₁₄) into the backbone of the polymer (see Byrom, 1994supra). A list of monomers and functional groups that have been found inmicrobial PHA is disclosed in Table 2.5 of Byrom (1994). Typically, thecomposition of the resulting PHA depends upon the growth substrate used(Madison and Huisman, 1999, supra). While the loci encoding PHAsynthesis genes have been characterized for at least 18 differentspecies, PHA biosynthesis and its coordinated gene expression inresponse to environmental conditions remains unclear and represents acomplex area under current investigation.

Quantitation of PHA Levels

PHA levels within a sample can be determined or measured for example, byremoving an aliquot of the biomass from the reactor and determining itsdry weight. Briefly, the biomass is lysed by addition of 0.1 mL ofconcentrated hydrochloric acid and 0.4 mL of n-propanol, and heattreatment at 100° C. for 2 hours. During this lysis step,polyhydroxyalkonoates are hydrolyzed and esterified. The esterifiedcompounds can then be quantitated by gas chromatography (GC) asdescribed by Riis and Mai, (Riis and Mai (1988) supra) and the dryweight percent PHA calculated.

PHA levels can also be determined using an automated fluorescence basedPHA method based upon that described by Ostle and Holt, 1982 (Appliedand Environmental Microbiology, 44:23) and modified as described below.Briefly, one milliliter of biomass is transferred from the bioreactor toa chamber into which is added 1 mL of Nile Blue (1%). The mixture isincubated at 55° C. for 15 min. The sample is washed with deionizedwater (or some suitably “clean” non-fluorescent liquid) to remove theexcess Nile Blue dye followed by an 8% acetic acid wash. The stainedbiomass is transferred to a cuvette/flow cell in a fluorescencespectrophotometer with the excitation wavelength set to 362 nm. Theamount of PHA present in the biomass is determined by comparison to astandard curve generated using either neat PHA polymer or biomasscontaining PHA that has been quantitated using the GC-FID (flameionization detector) method.

High levels of PHA in the biomass indicate that carbon flow in the cellhas been directed toward PHA formation and is an indicator of reducedbioreactor health. PHA production and denitrification compete forreduced forms of nicatinamide deoxyribose [NAD(P)H]. Therefore, PHAformation can be viewed as a non-productive side reaction reducing theperformance of the system for the desired reaction, i.e. reduction ofnitrate.

Specifically, Applicants have determined that when PHA accumulates tohigh levels in the biomass (about 15 to about 20% dry weight), thenutilization of exogenously supplied carbon (methanol and organic acids)slows. Appropriate adjustment of the feed nutrients to the bioreactorcan then be made to return the bioreactor to an efficient operatingcondition. Applicants' observation that high levels of PHA in thebiomass correlate to reduced denitrification activity can be used tomore rapidly assess the productivity of anoxic wastewater reactors.Since the build up of PHA in bacteria is primarily driven by nutrientlimitation (sulfate, sulfide, phosphate, and a nitrogen source, such asnitrate, ammonia, and/or urea), the flux of carbon into PHA can beeasily controlled by monitoring key nutrient levels in the effluent.

Wastewater Process Monitoring and Adjustments in Response to IncreasedPHA Level

The overall goal of wastewater systems is to reliably and efficientlytreat inlet streams (municipal or industrial) to convert organic carbonto carbon dioxide, and in some cases, organic and inorganic nitrogen togaseous end products. The general strategy is to use monitoring ofnutrient and carbon consumption along with PHA levels in the biomass asa gauge of system productivity to ensure performance of the bioreactor.

An indication that the biocatalytic efficiency of the wastewatertreatment process is impaired is seen when the polyhydroxyalkanoatesconcentration is greater than about 15 to about 20 dry weight percent ofthe biomass. Appropriate adjustment of the feed nutrients to thebioreactor can then be made to return the bioreactor to an efficientoperating condition. Preferably, the feed nutrients are adjustedaccordingly in the activated sludge environment when the PHAconcentration is from about 10 to about 20 dry weight percent of thebiomass. More preferably, the feed nutrients are adjusted accordingly inthe activated sludge environment when the PHA concentration is fromabout 10 to about 15 dry weight percent of the biomass.

Maintaining Viable Cultures in a Bioreactor During Reduced or AbsentCarbon Influx

The present invention also provides the means to maintain an activatedsludge system in the absence of carbon influx. Specifically, applicantshave developed a method for maintaining biological activity in theabsence of carbon influx to the bioreactor. This method is particularlyuseful during periods of reduced wastewater production, specificallyduring scheduled shut downs of the manufacturing process, which normallysupplies the carbon feed to the bioreactor. Currently, the only recourseto maintain viable biomass within the reactor is to supply it withpurchased carbon sources, such as corn steep liquor, molasses, oranother cheap carbon source. By understanding the mechanisms thatpromote PHA accumulation and providing methods to easily monitor andcontrol the amount of PHA present in the biomass, these polymers can beused as internally supplied carbon sources. The ability of most naturalbacteria to break down stored carbon for use as a carbon and energysource during periods of starvation is exploited in this method tomaintain biological activity in the wastewater bioreactor without theneed to purchase carbon sources.

Prior to a scheduled shut down, nutrient limitations can be used toshift the microbial physiology to PHA synthesis. Applicants have shownin Example 3 below that internal PHA is able to function as a carbonsource for the biomass during periods of process waste outages. PHAformation can be stimulated by limiting the amount of nitrogen,phosphorous, and/or sulfur sources that is supplied to the biomass. In aspecific embodiment, PHA formation can be stimulated by limiting theamount of nitrogen that is supplied to the biomass. The nitrogen sourcemay include but is not limited to ammonia, nitrate, and/or urea.Preferably, nutrient limitation of the biomass will begin from about 8weeks before to about 2 weeks before shut down. More preferably,nutrient limitation of the biomass will begin from about 8 weeks beforeto about 6 weeks before shut down. Even more preferably, nutrientlimitation of the biomass will begin at about 8 weeks before shut down.The biologically required amount of nutrients can be determined from thegrowth yield data, carbon load to the reactor and the amount of eachnutrient present in biomass (Wastewater Engineering: Treatment,Disposal, and Reuse. Metcalf and Eddy, Inc. 3^(rd) Edition. McGraw-Hill,Inc., New York, 1991).

During the shut down period, the level of PHA in the system should befollowed to insure that the biomass does not starve. Preferably, thelevel of PHA in the system will be at least than about 1% to about 5% ofthe MLSS. More preferably, the level of PHA in the system will be atleast than about 3% to about 5% of the MLSS. Even more preferably, thelevel of PHA in the system will be at least than about 5% of the MLSS.If the PHA level approaches about 1% to 2% of the MLSS, then purchasedcarbon sources can be supplied to keep the bioreactor viable untilwastewater carbon influx is resumed.

EXAMPLES

The present invention is further defined in the following non-limitingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usesand conditions.

General Methods

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,DC. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989).

Methods for analysis and determination of components present inwastewater bioreactors can be found in Eaton et al. (Standard Methodsfor the Examination of Water and Wastewater, 19^(th) edition, AmericanPublic Health Association, Washington, D.C., 1995).

All reagents, restriction enzymes and materials used for the growth andmaintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

The meaning of abbreviations is as follows: “h” means hour(s), “min”means minute(s), “sec” means second(s), “d” means day(s), “mg” meansmilligram, “g” means gram, “uL” means microliters, “mL” meansmilliliters, “L” means liters, “uM” means micromolar, “mM” meansmillimolar, “M” means molar, and “ppm” means parts per million.

Bioreactor Operation:

A modified Eckenfelder reactor was operated in continuous mode. Themodifications included a water jacket for temperature control and a headplate. The reactor volume was 280 mL and contained an internal settlingzone of 50 mL for a total volume of 330 mL, the temperature wasmaintained at 35° C. and the pH controlled to 7.50+/−0.15. The pH wasmaintained by addition of 0.1 M NaOH. The flow rate was set to 0.12mL/min establishing a hydrolic residence time of 1.9 days. The sludgeresidence time (sludge age) was 20 days. The sludge age was controlledthrough daily wasting of biomass from the system. Biomass wasting wasperformed by mixing the contents of the settling and reaction zones andremoving {fraction (1/20)}th of the reactor volume (20 mL/d). The massof bacteria present in the reactor was determined daily as follows:three milliliters of biomass was filter through a dried, preweighed 1.2μM glass filter (Gelman, Ann Arbor, Mich.), the biomass was dried for 1hour at 105° C. (dry weight) and combusted at 550° C. for 20 minutes(ashe weight). The oxidation reduction potential (ORP), pH, andtemperature were continuously logged to a computer through a dataacquisition system (IO Tech multiscan with DuPont Scan 1200 V1.3.0software).

Media Formulation:

The feed to the reactor was prepared in sterile deionized water. Nitricacid was added to bring the concentration of nitrate to 13.5 g/L. Twocompositions of organics were used: 1) methanol and 2) a combination ofmethanol, valeric acid and butyric acid. The methanol feed contained 9.0g/L and the mixture contained 4.5 g/L methanol, 1.79 g/l valeric acidand 1.96 g/L butyric acid. The pH was adjusted in both feed compositionsto approximately 1.3 by the addition of 2 mL of 50% sodium hydroxide.

Nutrient Addition:

The reactor was batch fed (75 μL/d) of the following nutrient solutions:Solution 1: comprising Na₂SO4, 82.0 g/L; KH₂PO₄, 65.6 g/L; H₃BO₃, 1.1g/L; NaMoO₄, and 0.5 g/L, NiCl 6H₂O; Solution 2 comprising F₃Cl 4H₂O,13.5 g/L); Solution 3 (MnCl 4H₂O, 7.0 g/L; CaCl₂ 2H₂O, 63.3 g/L; MgCl₂6H₂O, 110.3 g/L; CuCl₂ 2H₂O, 0.5 g/L; CoCl₂ 6H₂O, 0.8 g/L). Duringdefined periods, ammonia was also added to the system to a finalconcentration that ranged from about 99 to about 396 mg/L. The amountsof phosphate, sulfate and ammonia were determined by ion chromatography(see below).

Analytical Techniques

COD Analysis:

The chemical oxygen demand (COD) of the feed and in the bioreactor wasmeasured using Hach COD (Hach Corp., Loveland Colo.) vials following themanufacturer's protocol.

Methanol Analysis:

The concentration of methanol in the reactor was determined by GC/FID asdescribed above (Riis and Mai (1988) supra). Briefly, biomass wasremoved from the reactor and centrifuged at 13,000 rpm in a microfuge(Heraeus Instruments, USA) at4° C. The supernatant was filtered througha 0.2 micron filter (Gelman). An HP 6890 GC Hewlet Packard instrumentcontaining a HP-5 capillary column was operated using the followingparameters: inlet temperature of 250° C., a helium carrier gas flow rateat 1.5 mL/min, a column temperature initially set at 60° C. and rampedto 250° C. at 25° C./min, an FID temperature of 250° C. and a hydrogengas flow rate of 40 mL/min, an air flow rate of 450 mL/min, and a heliumflow rate of 45 mL/min. An injection volume of 2 uL was used with a 20:1split at the inlet. Cyclopentanone was used as the internal standard anda linear calibration curve for methanol concentrations ranging from 0 to1000 mg/l (r² 0.99) was used to determine reactor methanolconcentration.

Anion Analysis:

The concentrations of valeric acid, butyric acid, nitrate, and nitritewere determined by Ion Chromotography using a Dionex IC System. Anionswere analyzed using the Dionex ion chromatography isocratic method foranion analysis. Briefly, AS11-HC analytical columns with AS11-HC guardwere used with a pump flow rate of 1.5 mL/min. The Detector wasconductivity and the eluent was 24 mM NaOH. Sample runtime was 16minutes using an injection volume of 25 μL. The primary anions analyzedwere chloride, nitrite, sulfate, nitrate, and phosphate. Externalstandards were used to generate standard curves. Standard concentrationsof 0 mg/L, 1 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, and 50 mg/L with r²>0.99for each component were used to generate the standard curves. Sampleswere taken from the reactor at different times and centrifuged in 1.5 mLmicro-centrifuge tubes for 5 min at 13,000 rpm. The supernatant wasdiluted 1:5 with deionized water and placed on the autosampler foranalysis.

Cation Analysis:

Cations were analyzed using the Dionex ion chromatography isocraticmethod for cation analysis. Briefly, CS12A analytical columns with CS12Gguard were used with a pump flow rate of 1.0 mL/min. The detector wasconductivity and the eluent was 22 mN H₂SO₄. Sample run time was 16 minusing an injection volume of 15 μL. The primary cations analyzed weresodium, ammonium, potassium, magnesium, and calcium. External standardswere used to generate a standard curve. Standard concentrations of 0mg/L, 5 mg/L, 10 mg/L and 25 mg/L, with r²>0.99 for each component wereused to generate the standard curves. Samples were taken from thereactor at different times and centrifuged in 1.5 mL micro centrifugetubes for 5 min at 13,000 rpm. One mL of the sample's supernatant wasthe placed into a GC screw top vial containing 10 μL of H₂SO₄ and eitherplaced in the refrigerator at 4° C. or diluted 1:5 with deionized waterand placed on the autosampler for analysis.

Nitrous Oxide Analysis:

The dissolved nitrous oxide concentration was determined by thefollowing method. Briefly, a 1 mL reactor sample was placed into a 2.0mL crimp cap vial and the sample was acidifed by adding 10 μL ofconcentrated HCL to stop biological activity. Samples were stored at 4°C. until analyzed. Samples were heated overnight at 80° C. to establishequilibrium between the dissolved and gaseous nitrous oxide. Nitrousoxide levels were determined by GC/ECD (HP instruments) using a Supelco80/100 Porapak Qss column. The inlet and column temperatures were 30° C.and the total gas flow rate was 30 mL/min (5% methane, 95% Argon). Theinjection was 100 μL of the vial head space and the GC was operated insplitless mode. Nitrous oxide concentration was determined from anexternal standard curve made using gas standards. 0, 10, and 100 ppm N₂0(Roberts Gas, Co.).

PHA Concentration Determination:

Biomass was removed from the reactor and transferred to a preweighed 7mL vial and centifuged at 4000 rpm for 10 min in a Sorval SS34 rotor at4° C. The supernatant was removed and the biomass pellet is dried at105° C. overnight. The dry weight of the biomass was determined. Themass of bacteria present in the reactor was determined daily as follows:three milliliters of biomass was filter through a dried, preweighed 1.2μM glass filter (Gelman), the biomass was dried for 1 hr at 105° C. (dryweight) and combusted at 550° C. for 20 minutes (ashe weight).

The common PHB method is derived from the method developed by Riis andMai (Riis and Mai (1988) supra).

The method is based on the hydrolysis and transesterification of PHBwith propanol and hydrochloric acid to hydroxybutryric acid propylester. Benzoic acid is used as the internal standard. Briefly, thismethod involves adding 1 mL of Dichloroethane (DCE), 200 μL of Benzoicacid stock solution (2.0 g Benzoic acid in 50 mL of 1-propanol), and 1mL of a Propanol-HCl stock solution (1 volume concentrated hydrochloricacid and 4 volumes 1-propanol) to the vial containing the dry biomasssample. The re-suspended sample is then reacted in a heat block for 2hours at 100° C. with periodic shaking. The sample is then cooled toroom temperature and the reaction is quenched by adding 2 mL ofdeionized water. Five μL of the organic phase (lower phase) is removedand injected into an HP 6890 GC equipped with a HP-5 5% PhenylMethyl-Siloxane capillary column. The GC analysis parameters used were agas flow rate of 1.5 mL/min, injection made with the inlet-split mode,with a split ratio=100:1, an inlet temperature of 180° C. with helium asthe carrier gas. The column temperature was initially 100° C. and at theend of the method was 225° C. using a 25° C./min ramp rate. The columnwas held at 100° C. for 0.5 min, and the total run time was 7.0 min. Thedetector was a flame ionization detector (FID) operated at 275° C. Thegas flow rate was 40 mL/min (Hydrogen) and the air flow rate was 450mL/min with a Helium make up flow of 50 mL/min.

PHA Calibration Standards Preparation Procedure:

The PHA concentration is determined by comparison to standard containingknown amounts of PHBA and PHVA (Sigma Chemical company). A 20 g/LPHB/PHV stock solution was prepared by dissolving 100 mg of PHB/PHV in 5mL dichloroethane (DCE) and heating at 100° C. The vessel comprisingthis stock solution should be a calibrated sealed vial. After thePHB/PHV goes into solution, it was cooled to room temperature and DCEwas added to restore the original volume. This 20 g/L PHB/PHV stocksolution and dilutions prepared from it can be used to detect a range ofreactor biomass PHA concentrations from 0 mg/ml to about 16.4 mg/ml. Themaximum amount of biomass used was approximately 15 mg, therefore, theamount of PHA in the reactor biomass was within the range of the PHAconcentrations provided by the standards.

PHA concentration can also be determined using the automatedfluorescence method based upon Ostle and Holt (1982) and modified asdescribed above in the Detailed Description of the Invention.

Determination of Denitrification Potential:

Denitrification potential, the rate of reduction of nitrate to nitrogenper unit of biomass, was determined using biomass from an anoxicbioreactor. The biomass was removed from the reactor and transferred toa centrifuge tube, pelleted and washed with a phosphate buffer solution(pH 7.5, 50 mM), and resuspended in an S12 mineral salts media (10 mMNH₄SO₄; 50 mM KPO₄, pH 7.0; 2 mM mgCl₂; 0.7 mM CaCl₂; 0.05 mM MnCl₂;0.001 mM FeCl₃; 0.001 mM ZnCl₃; 1.72 μM CuSO₄; 2.53 μM COCl₂; and 2.42μM NaMoO₂) and sparged with argon to remove oxygen. The vials containedbetween 400 to 500 mg/L of reactor biomass. Various carbon mixes and 20mg/L nitrate-N (wherein 20 mg/L nitrate-N multiplied by 4.5 g NO₃/gN=90mg/L nitrate) were added into anaerobic vials to initiate the reaction.The carbon sources used consisted of the feed composition (50/50methanol and organic acids), methanol, organic acids (valeric andbutyric) and a no carbon control. The reaction using the feedcomposition contained 33 mg/L methanol, 12.3 mg/L valeric acid and 13.6mg/L butyric acid. The methanol reaction contained 66 mg/L methanol andthe organic acids reaction contained 24.5 mg/L valeric acid and 27.2mg/L butyric acid. The rates of nitrate removal and carbon consumptionwere measured as a function of time using the ion chromatography methoddescribed above. The studies were performed at 35° C. and pH 7.5, theoperating conditions of the anoxic reactor.

Example 1 Correlation Between PHA and Denitrification in IsolatedCultures

This example describes the impact of feed composition on denitrificationrates as determined in a continuous anoxic bioreactor. The firstoperating condition comprised a feed composition comprising 90%methanol, 5% adipic acid, 2.5% cyclopentanone, and 2.5%hexamethylenediamine, referred to herein as “methanol rich feed”. Thesystem was operated in this regime for several sludge ages, i.e., equalto the amount of time needed to replace all of the biomass present inthe reactor, so that the operating performance and systemcharacteristics using the methanol rich feed could be determined.Following this period, the system was switched to a feed compositioncomprising 50% methanol, 25% butyric acid and 25% valeric acid, referredto as “organic acid rich feed” (Arrow 1 on FIGS. 1 and 2). The systemwas maintained on this feed for several sludge ages and characterized atthe macroscopic level using measurements of carbon, nitrate/nitrite, andbiomass (MLVSS and MLSS). At various times during this operating period,batch studies were used to measure the denitrification uptake rate (NUR)on various carbon sources. The carbon sources used consisted of the feedcomposition (50/50 methanol and organic acids), methanol, organic acids(valeric and butyric acids), and a no carbon control.

The macroscopic performance of the methanol rich feed system is shown inFIG. 1 and Table 1.

TABLE 1 Reactor Performance without Ammonia Addition Feed NO₃ LoadReactor NO₃ Effluent NO₃ Day mg/day mg/L mg/L 1 2246.0 8.0000 0.0000 22184.0 0.0000 0.0000 5 2278.0 8.0000 8.0000 8 2278.0 ND ND 12 1917.0 NDND 25 1874.0 0.0000 13.000 32 1642.0 0.0000 0.0000 39 2421.0 0.00000.0000 43 2421.0 ND 0.0000 46 2328.0 0.0000 0.0000 53 2384.0 0.000017.000 60 2233.0 0.0000 0.0000 62 2213.0 183.00 ND 64 2213.0 4.0000 ND67 2182.0 0.0000 0.0000 71 2223.0 0.0000 ND 74 ND 0.0000 ND 75 2286.0491.00 8.0000 76 ND 0.0000 ND 81 2178.0 0.0000 0.0000 82 2050.0 ND ND 832060.0 40.000 0.0000 88 2203.0 0.0000 0.0000 90 1851.0 104.00 0.0000 911194.0 0.0000 0.0000 92 1191.0 8.0000 8.0000 93 0.0000 ND ND 95 2401.0ND 0.0000 96 ND 83.000 ND 97 2401.0 ND ND 98 ND 0.0000 ND 99 2286.00.0000 ND 101 ND 0.0000 ND 102 2300.0 8.0000 8.0000 104 2181.0 8.00000.0000 106 2170.0 0.0000 0.0000 109 2250.0 8.0000 0.0000 111 2151.046.000 8.0000 113 2276.0 29.000 0.0000 116 2294.0 103.00 0.0000 1182143.0 94.000 8.0000 120 2149.0 60.000 0.0000 123 2222.0 0.0000 0.0000125 2447.0 0.0000 0.0000 127 2438.0 866.00 0.0000 130 1865.0 0.00000.0000 132 1782.0 81.000 0.0000 133 ND 346.00 ND 134 1747.0 500.000.0000 137 2038.0 129.00 0.0000 139 ND 1641.0 ND 140 1905.0 865.0038.000 143 1848.0 81.000 0.0000 146 ND 433.00 ND 147 ND ND ND 148 1118.01224.0 0.0000 151 1476.0 0.0000 0.0000 153 1776.0 170.00 0.0000 15421.000 ND ND 155 16.000 14.000 0.0000 158 11.000 16.000 0.0000 1601065.0 19.000 12.000 165 ND 180.00 0.0000 166 236.00 264.00 0.0000 167ND 0.0000 ND 168 102.00 74.000 ND 169 114.00 37.000 0.0000 172 117.000.0000 0.0000 173 155.00 144.00 0.0000 174 554.00 17.000 ND 175 1044.00.0000 0.0000

Operation on the methanol rich feed composition resulted in robustperformance as characterized by no nitrite or low residual nitratelevels in the effluent. Efficiency of denitrification droppeddramatically following the shift to the organic acid rich feedcomposition (Arrow 1, FIG. 1). The nitrate load to the system wasreduced to zero to simulate shut down two times (Arrows 2 and 3, FIG. 1)and residual carbon and nitrate were flushed from the reactor using a 5mM phosphate buffer, pH 7.5.

The impact of these operating conditions on NUR using various carbonsources and the corresponding reactor PHA levels is shown in FIG. 2.Operation with the methanol rich feed resulted in consistent NUR valuesin the range 3.35×10⁻⁴+/−0.2×10⁻⁴ mg NO₃-N/mg MLVSS-min with methanol asthe carbon source (FIG. 2A). This denitrification rate is sufficient toensure complete reduction of the feed nitrate. Following a shift to theorganic acid rich feed (Arrow 1 on FIG. 2A), a significant reduction inthe rate of denitrification was observed. The NUR with methanol as thecarbon source decreased 10 fold within 21 days following the feed shift(FIG. 2B). Also during this period, the endogenous rate increased by 3fold as determined in the no carbon control shown in FIG. 2C. Theincrease in the endogenous rate indicates that the biomass was using aninternally stored carbon source. However, the NUR with the feedcomposition was reduced (approximately 3 fold) during in the same timeframe (21 days) as shown in FIG. 2A. These data indicate a generalreduction in the specific NUR as a function of time following the switchfrom methanol rich feed to a feed comprising both methanol and organicacids and referred to as the organic acid rich feed. As mentioned above,the reactor was not able to continually metabolize the nitrate andcarbon at the rate at which it was being fed. The feed was then switchedto a phosphate buffer (pH 7.5) for 24 hours to flush the residual carbonand nitrate from the reactor (Arrow 2 on FIG. 2A). The system wasrestarted on the organic acid rich feed. The NUR on the organic acidrich feed composition was reduced by approximately 2 fold with the lossof denitrification performance observed within a similar time frame,approximately 21 days (see FIG. 2A) as compared to the first period ofoperation. The NUR with methanol as the carbon source decreased by afactor of 10 (FIG. 2B). Also consistent with the first period ofoperation, the endogenous NUR of the second operation increased by afactor of 1.5 (FIG. 2C).

The loss of nitrate reduction performance shown in FIG. 1 (approximatelyday 95) was reproducible. This behavior was repeated by operating thereactor under the same conditions that resulted in loss of nitratemetabolism. These data are shown in FIG. 1 from 98 to 155 days (betweenArrows 2 and 3). The macroscopic characteristics of the system were thesame, i.e. reduced nitrate reduction rates and increased levels of PHAin the biomass as shown in FIG. 2. Note the arrow numbers in FIGS. 1 and2 refer to the same operating periods. These data indicate that the lossof denitrification (nitrate reduction) performance is reproducible anddriven by a decreased ability of the biomass to use the exogenouslysupplied carbon to drive denitrification as indicated by an increasedNUR in the no carbon added sample (FIG. 2C). Applicants have hereindetermined that the internal carbon source present in the biomass is PHA(see FIG. 2D).

The correlation between NUR and PHA content in the biomass is shown inFIG. 3 and Table 2. The batch NUR measured with the organic acid richfeed composition was reduced by a factor of 3-4 at PHA levels above15-20%. The data presented in FIG. 3 and Table 2 are a composite of allof the data collected during the various operating conditions.

TABLE 2 Percent maximum specific nitrate uptake rates and percent PHA %Maximum % Maximum SNUR SNUR % MeOH Feed PHA 100.0 143.0 0.0 125.0 100.01.0 75.0 84.0 1.0 46.0 49.0 2.0 94.0 93.0 6.0 63.0 92.0 6.0 66.0 86.07.0 64.0 96.0 8.0 58.0 84.0 11.0 64.0 55.0 15.0 100.0 63.0 17.0 80.086.0 18.0 4.0 17.0 24.0 3.0 23.0 24.0 2.0 16.0 25.0 7.0 28.0 26.0 7.030.0 28.0 7.0 20.0 28.0 9.0 22.0 30.0

Although, some of the data fall outside of the general trend, themajority is consistent with the observation that increased PHA levels inthe biomass resulted in reduced nitrate uptake rates. With methanol asthe carbon source, the NUR was reduced approximately by an order ofmagnitude at PHA levels above 20%.

The impact of PHA levels on system performance can be determined bycomparing the system's nitrate load, which is equal to [feed nitrate (mgNO₃-N/L)]×[flow rate (L/min)], to the denitrification capacity in thereactor. The system capacity can be estimated by multiplying the maximumNUR by a PHA inhibition term and the total amount of biomass in thesystem using Equation 2.

System capacity=[Maximum nitrate rate (mg nitrate-N/mgMLVSS-min)−reduction due to PHA]×[Total biomass in reactor (mgMLVSS)].  EQUATION 2:

The term for the reduction in NUR caused by PHA is determined by linearregression analysis of the data shown in FIG. 3 as shown in Equation 3.

NUR reduction=(1.5×10⁻⁵ mg nitrate-N/mgMLVSS-min-%PHA)×(%PHA).  EQUATION 3:

The system capacity must be greater than or equal to the nitrate load tothe system for complete denitrification to occur. Therefore, Applicants'invention provides one of ordinary skill in the art the ability tomonitor PHA levels within the biomass of a wastewater bioreactor andpredict the denitrification capacity in the reactor to determine systemperformance. Appropriate adjustments to the influx, such as nutrientcontrol, can be made similar to that described in Example 2 below tomaintain efficient system performance.

Example 2 Effect of the Addition of Various Feed Nutrients on PHA Levels

Example 2 demonstrates that denitrification performance is impacted byPHA level in the biomass. Specifically, an increase in PHA contentcorresponded to a diminished ability of the biomass to use methanol as acarbon source to drive denitrification (see FIGS. 2 and 3 and Table 2).Accumulation of PHA is a physiological response to excess carbon andnutrient starvation, and can be minimized by the addition of macro- andmicronutrients such as ammonia, sulfate, and phosphate. A reactor feedcomposition was chosen that contained excess ammonia (1.5 times thephysiologically required amount) to minimize PHA accumulation andpromote reliable denitrification performance.

To test the ability of ammonia to control PHA levels in the biomass, theorganic acid rich feed composition described in Example 1 was used sincePHA levels were higher with this feed composition. The macroscopicperformance of the system is shown in FIG. 4. During operation withexcess ammonia, the system performance was very stable and reliable andno nitrate leaks were observed. Performance under these reactorconditions contrasts significantly to operation without the addition ofammonia as described in Example 1 (see FIG. 1).

NUR experiments were conducted by using biomass from the anoxicbioreactor as described above in Example 1. The nitrate uptake rateusing various carbon sources and the corresponding reactor PHA levelsare shown in FIG. 5. The NUR with the organic acid rich feed compositionas the carbon source with ammonia shown in FIG. 5A resulted in moreconsistent nitrate removal rates, ranging from 2.82×10⁻⁴ to 6.75×10⁻⁴ mgnitrate-N/mg MLVSS-min. The average feed NUR (organic acid rich feedcomposition) was approximately 30% higher with ammonia addition thanwithout ammonia addition (compare FIGS. 5A to 2A, respectively). Therate of nitrate removal with methanol as the carbon source ranged from1.49×10⁻⁴ to 3.24×10⁻⁴ mg nitrate-N/mg MLVSS-min as shown in FIG. 5B.These rates are comparable to the maximum NUR observed during theprevious operating regimes (methanol rich feed and organic acid richfeed, both without ammonia addition, FIGS. 1 and 2). The relativelyuniform NUR during this period (FIG. 5B) is a sharp contrast to thehighly variable NUR rate (methanol as the carbon source) observed duringoperation with organic acid rich feed without nutrient control (FIG.2B).

The concentration of PHA in the biomass as a function of operatingcondition is shown in FIG. 5D. The levels of PHA were approximately twofold higher in the absence of ammonia addition (nutrient control, seeFIG. 2D) as compared to operation with nutrient control (FIG. 5D).Without nutrient control, a large fraction (15 to 38%) of thebiocatalyst (biomass) is inert polyester (PHA) (FIG. 2D). In contrast,nutrient addition controls the amount of PHA to about 10% or less (FIG.5D). These results are consistent with the low endogenous NUR observedin the no carbon control reactions as shown in FIG. 5C.

Addition of excess ammonia resulted in reduced levels of PHA in thebiomass and consistent nitrate uptake rates. The nitrate utilizationrate was approximately 30% higher than the maximum NUR observed in thetwo previous operating regimes (methanol rich, and organic acid rich,both without ammonia). These results highlight the impact of operatingconditions on the performance of an anoxic system and the need tomonitor both biomass composition and levels in wastewater bioreactors.Addition of excess ammonia resulted in stable and reliabledenitrification performance and reduced levels of PHA in the biomass.

Therefore, monitoring of PHA and nutrient levels in wastewater treatmentsystems can be used to gauge the denitrification potential and as ageneral measure of system health. An indication that the biocatalyticefficiency of the wastewater treatment process is impaired is seen whenthe polyhydroxyalkanoates concentration is greater than about 15 toabout 20 dry weight percent of the biomass. Appropriate adjustment ofthe feed nutrients to the bioreactor can then be made to return thebioreactor to an efficient operating condition. Preferably, the feednutrients are adjusted accordingly in the activated sludge environmentwhen the PHA concentration is from about 10 to about 20 dry weightpercent of the biomass. More preferably, the feed nutrients are adjustedaccordingly in the activated sludge environment when the PHAconcentration is from about 10 to about 15 dry weight percent of thebiomass. Adjustments made to the feed nutrients (i.e., addition ofexcess ammonia, nitrate, urea, phosphorous sources, sulfur sources, andthe like) are effective to achieve optimum health and denitrificationability of the bioreactor.

Example 3 An Activated Sludge System in the Absence Of Carbon Influx

This example describes a method for maintaining biological activityduring periods of reduced or absent carbon influx to the bioreactor.This method is useful during periods of scheduled shut downs of themanufacturing process, which normally supplies the carbon feed to thebioreactor, or any other time of significantly reduced carbon influx.Currently, the only recourse to maintain viable bioreactor culturesduring a shut-down period is to supply purchased carbon sources such ascorn steep liquor, molasses, or another cheap carbon source to thebioreactor. Applicants have identified the conditions under which PHAaccumulation can be promoted and provide methods to easily monitor andcontrol the amount of PHA present in the biomass. Since these polymerscan be used as internally supplied carbon sources, PHA accumulation canbe achieved prior to the scheduled shut down or low carbon influxperiod. The ability of most natural bacteria to break down the storedPHA for use as a carbon and energy source during periods of starvationcan then be exploited in a method to maintain biological activity in thewastewater bioreactor without the need to purchase carbon sources.

Applicants have demonstrated herein that nutrient limitations can beused to shift the microbial physiology to PHA synthesis prior to ascheduled shut down. Specifically, PHA formation can be stimulated bylimiting the amount of nitrogen, phosphate, or sulfate that is suppliedto the biomass. The biologically required amount of nutrients can bedetermined from the growth yield data, carbon load to the reactor andthe amount of each nutrient present in biomass (Wastewater Engineering:Treatment, Disposal, and Reuse. Metcalf and Eddy, Inc. 3^(rd) Edition.McGraw-Hill, Inc., New York, 1991).

Briefly, ammonia addition was gradually withdrawn, starting at about 8weeks prior to the scheduled shut down. The PHA levels were monitoredwithin the biomass on a 1-2 day basis until PHA levels reachedapproximately 20% dry weight of the biomass. The carbon influx wasstopped and the PHA levels, MLVSS, and nitrate metabolism were monitoredusing the methods as described above. The amount of PHA in the biomassdropped from 20% to about 2% in one week and a total of 121 mg ofnitrate were metabolized (see FIG. 6).

As shown in FIG. 7, PHA began to build up in the biomass approximately30 days after ammonia addition ceased. Within approximately 55-60 days(approximately 8 weeks), PHA levels range between 15 and 40 weightpercent. As shown in FIG. 6, approximately 20% (PHA wt/biomass wt) PHAwas sufficient to support maintenance of the biomass for 6-7 days.Therefore, prior to a scheduled shut down, it is preferable to initiatenutrient limitation about 8 weeks in advance of the shut down to inducePHA accumulation.

These results demonstrate that internal PHA can function as a carbonsource during periods of process waste outages, provided that the levelof PHA in the system is followed to insure that the biomass does notstarve. Preferably, the level of PHA in the system will be at least thanabout 1% to about 5% of the MLSS. More preferably, the level of PHA inthe system will be at least than about 3% to about 5% of the MLSS. Evenmore preferably, the level of PHA in the system will be at least thanabout 5% of the MLSS. If the PHA level approaches about 1% to 2% of theMLSS, then purchased carbon sources can be supplied to keep thebioreactor viable until wastewater carbon influx is resumed.

What is claimed is:
 1. A method for controlling the biocatalyticefficiency of a wastewater treatment process comprising: a) providing anactivated sludge environment comprising: (i) a carbon influx; (ii)cultures of autotrophic, heterotrophic and facultative microorganisms;(iii) a feed nutrient; and (iv) an end electron acceptor b) samplingwastewater from anaerobic, anoxic and/or aerobic stages of the treatmentprocess; c) measuring the concentration of an internal storage moleculeselected from the group consisting of polyhydroxyalkanoates andglycogen, which is present in the sample, to determine the status of aselected sample characteristic; and d) adjusting the feed nutrient inthe activated sludge environment depending on the status of the selectedsample wherein the biocatalytic efficiency of a wastewater treatmentprocess is controlled.
 2. The method according to claim 1, wherein whenthe concentration of polyhydroxyalkanoates is greater than about 15 toabout 20 dry weight percent of the biomass, there is an indication thatthe biocatalytic efficiency of the wastewater treatment process isimpaired.
 3. The method according to claim 1, wherein the sampling is insitu.
 4. The method according to claim 1, wherein the sampling iscontinuous.
 5. The method according to claim 1, wherein the carboninflux comprises a compound selected from the group consisting of,amines, alcohols, organic acids, carbohydrates, proteins, and aminoacids.
 6. The method according to claim 1, wherein the cultures ofautotrophic, heterotrophic and facultative microorganisms compriseorganisms selected from the group consisting of alpha, beta, and gammaProteobacteria.
 7. The method according to claim 6, wherein the alpha,beta, and gamma Proteobacteria are selected from the genera consistingof Paracoccus, Rhodococcus, Pseudomonas, Alcaligenes, Acinetobacter,Sphingamonas, Azoarcus, and Burkholderia.
 8. The method according toclaim 1, wherein the feed nutrient is selected from the group consistingof nitrate, ammonia, sulfate, sulfide, urea and phosphate.
 9. The methodaccording to claim 1, wherein the end electron acceptor is selected fromthe group consisting of oxygen, nitrate, nitrite, nitrous oxide, ferricoxide, and sulfate.
 10. The method according to claim 1, wherein thepolyhydroxyalkanoates are comprised of compounds selected from the groupconsisting of hydroxybutyrate and hydroxyvalerate.
 11. The methodaccording to claim 1, wherein the sample characteristic is selected fromthe group consisting of denitrification efficiency, nitrateconcentration, ammonia concentration, sulfate concentration, phosphateconcentration, and carbon dioxide concentration.